We previously described 4Dm2m, an exceptionally potent broadly neutralizing CD4-antibody fusion protein against HIV-1. It was generated by fusing the engineered single human CD4 domain mD1.22 to both the N and C termini of the human IgG1 heavy chain constant region and the engineered single human antibody domain m36.4, which targets the CD4-induced coreceptor binding site of the viral envelope glycoprotein, to the N terminus of the human antibody kappa light chain constant region via the (G4S)3 polypeptide linkers. This protein was further characterized 1) Protein- or peptide-based viral inactivators can be developed as novel antiviral drugs with improved efficacy, as well as improved pharmacokinetics and toxicity profiles, since they can actively attack and inactivate cell-free virions before they attach to host cells. In contrast, most clinically used antiviral drugs must penetrate host cells to inhibit viral replication for passive defense. We evaluated HIV inactivation activity of two gp120-targeting bispecific multivalent proteins, 2Dm2m and 4Dm2m, and three gp41-targeting HIV fusion inhibitory peptides, enfuvirtide (T20), T2635, and sifuvirtide (SFT), alone or in combination, by using PEG-6000 to separate HIV-1 virions from the antiretroviral drugs, followed by testing the residual infectivity of the HIV-1 virions. We found that both gp120-targeting proteins exhibited significant inactivation activity against all HIV-1 strains tested with EC50 values at low nM level, while none of the gp41-targeting peptides showed inactivation activity at concentrations up to 250 nM. However, combining one of the gp120-binding proteins with any single gp41-binding peptide exhibited potent synergistic inactivation activity against cell-free HIV-1 virions, including HIV-1 laboratory-adapted and primary HIV-1 strains, as well as those resistant to T20 or T2635 and those virions released from the reactivated latently HIV-infected cells with low combination index (CI) and significant dose reduction. These results indicate that the two gp120-targeting bispecific multivalent proteins, 2Dm2m and 4Dm2m, have potential for further development as HIV-inactivator-based antiviral drugs for use in clinics, either alone or in combination with gp41-targeting HIV fusion inhibitors, such as T20, for treating patients with HIV-1-infection/AIDS. 2) The envelope (Env) glycoprotein of HIV is the only intact viral protein expressed on the surface of both virions and infected cells. Env is the target of neutralizing antibodies (Abs) and has been the subject of intense study in efforts to produce HIV vaccines. Therapeutic anti-Env Abs can also exert antiviral effects via Fc-mediated effector mechanisms or as cytotoxic immunoconjugates, such as immunotoxins (ITs). In the course of screening mAbs for their ability to deliver cytotoxic agents to infected or Env-transfected cells, we noted disparities in their functional activities. Different mAbs showed diverse functions that did not correlate with each other. For example, mAbs against the external loop region of gp41 made the most effective ITs against infected cells but did not neutralize virus and bound only moderately to the same cells that they killed so effectively when they were used in ITs. There were also differences in IT-mediated killing among transfected and infected cell lines that were unrelated to the binding of the mAb to the target cells. Our studies of a well-characterized antigen demonstrate that mAbs against different epitopes have different functional activities and that the binding of one mAb can influence the interaction of other mAbs that bind elsewhere on the antigen. These results have implications for the use of mAbs and ITs to kill HIV-infected cells and eradicate persistent reservoirs of HIV infection. 3) We have previously generated an exceptionally potent and broadly neutralizing bispecific hexavalent protein, LSEVh-LS, containing four molecules of an engineered one-domainCD4, which binds gp120, and two molecules of an antibody domain targeting the HIV-1 co-receptor binding site fused to human IgG1 Fc. We defucosylated LSEVh-LS (LSEVh-LS-F) to improve its capacity to mediate antibody-dependent cell-mediatedular cytotoxicity (ADCC) activity and eliminate HIV-1-infected cells. In addition to evaluating the in vitro neutralization activity of LSEVh-LS-F, we investigated its in vivo anti-HIV-1 activity in humanized-mouse and non-human-primate experimental models. In both animal models, LSEVh-LS-F exhibited potent anti-HIV-1 activity. Using the humanized mouse model we demonstrated that LSEVh-LS-F was active against HIV-1 strains resistant to some potent broadly neutralizing antibodies (bNAbs) including VRC01 and 3BNC117. In both animal models, LSEVh-LS-F exhibited potent anti-HIV-1 activity. Using the humanized mouse model we demonstrated that in addition to its exceptionally potent HIV-1 neutralizing activity, LSEVh-LS-F also markedly reduced the number of infected cells through NK cell-mediated ADCC. However, as a consequence of its shorter half-life as compared to described mAbs, its potent anti-HIV activity was limited to several days after administration. Despite this disadvantage, the new inhibitor has the potential of neutralizing all isolates which use CD4 as a primary receptor, and exhibiting exceptionally potent NK cell-mediated ADCC activity capable of effectively eliminating HIV-1-infected cells. Therefore, LSEVh-LS-F represents a new potent agent which should contribute to the efficacy of combination treatments for depletion of the HIV-1 reservoir.